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SUMMARYThe Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy andin silicopredictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

Description, Novel macrocyclic peptides are found that bind and modulate the function of the retromer membrane trafficking complex., The retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signaling. Mutation of the retromer subunit Vps35 causes late-onset Parkinson’s disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function, we have created a novel series of macrocyclic peptides that bind retromer with high affinity and specificity. Crystal structures show that most of the cyclic peptides bind to Vps29 via a Pro-Leu–containing sequence, structurally mimicking known interactors such as TBC1D5 and blocking their interaction with retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting retromer. Last, tagged peptides can be used to probe the cellular localization of retromer and its functional interactions in cells, providing novel tools for studying retromer function.

The sorting nexin SNX17 controls endosomal recycling of transmembrane cargo proteins including integrins, the amyloid precursor protein, and lipoprotein receptors. This requires association with the Commander trafficking complex and depends on the C terminus of SNX17 through unknown mechanisms. Using proteomics, we find that the SNX17 C terminus is sufficient for Commander interaction and also associates with members of the PDZ and LIM domain (PDLIM) family. SNX17 contains a type III PDZ binding motif that binds specifically to the PDLIM proteins. The structure of the PDLIM7 PDZ domain bound to the SNX17 C terminus reveals an unconventional perpendicular peptide interaction mediated by electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/454387v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@137afd1org.highwire.dtl.DTLVardef@f7c91eorg.highwire.dtl.DTLVardef@1b4f2eborg.highwire.dtl.DTLVardef@b82f7c_HPS_FORMAT_FIGEXP M_FIG C_FIG

The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

The Retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signalling. Mutations in Retromer cause late-onset Parkinson’s disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function we have created a novel series of macrocyclic peptides that bind Retromer with high affinity and specificity. Crystal structures show the majority of cyclic peptides bind to Vps29 via a Pro-Leu-containing sequence, structurally mimicking known interactors such as TBC1D5, and blocking their interaction with Retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds Retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting Retromer. Finally, tagged peptides can be used to probe the cellular localisation of Retromer and its functional interactions in cells, providing novel tools for studying Retromer function.